Optimization of In vitro proliferation assay for haemonchus contortus antigen sensitized lymphocytes in goats

Abstract

The cell-mediated immune responses generated in the host could be measured by using in vitro lymphocyte proliferation assay. Once the lymphocytes are sensitized in vivo for a specific antigen, in vitro antigenic stimulation causes exponential antigen specific lymphocyte proliferation. Haemonchus contortus, an abomasal blood sucking parasite, induces cell-mediated immune responses in goats. Current study was conducted to optimize the methodology of in vitro proliferation at different antigen concentrations and to evaluate the cell proliferation using flow cytometer. Blood was collected from four goats (Saanen breed, aged less than 6 months) that were infected with H. contortus. Peripheral Blood Mononuclear Cells (PBMC) were isolated using the standard Pancoll- hypaque technique. Cells were cultured in complete RPMI media (RPMI 1640 supplemented with 2.05 mM L-glutamine, 2 g/L sodium bicarbonate, penicillin (100 IU/mL) and streptomycin (100 μg/ml) and 10% FBS) for 3 days at 37°C with 5% CO₂. Well known lymphocyte mitogen, Concanavalin A was used as a positive control at 5, 10 and 20 μg/mL, and H. contortus L3 somatic larval antigen was used at 200, 100, 50, 25, 10, 8, 6, 4 and 2 μg/mL concentrations. Concanavalin A at 5 μg/mL and the somatic larval antigen at 25 μg/mL yielded maximum in vitro lymphocyte proliferation. The results confirmed the basic requirements essential for goat in vivo sensitized lymphocyte proliferation.